Bacterial chemolithoautotrophy in ultramafic plumes along the Mid-Atlantic Ridge

Abstract Hydrothermal vent systems release reduced chemical compounds that act as an important energy source in the deep sea. Chemolithoautotrophic microbes inhabiting hydrothermal plumes oxidize these compounds, in particular, hydrogen and reduced sulfur, to obtain the energy required for CO2 fixation. Here, we analysed the planktonic communities of four hydrothermal systems located along the Mid-Atlantic Ridge: Irinovskoe, Semenov-2, Logatchev-1, and Ashadze-2, by combining long-read 16S rRNA gene analysis, fluorescence in situ hybridization, meta-omics, and thermodynamic calculations. Sulfurimonas and SUP05 dominated the microbial communities in these hydrothermal plumes. Investigation of Sulfurimonas and SUP05 MAGs, and their gene transcription in plumes indicated a niche partitioning driven by hydrogen and sulfur. In addition to sulfur and hydrogen oxidation, a novel SAR202 clade inhabiting the plume, here referred to as genus Carboxydicoccus, harbours the capability for CO oxidation and CO2 fixation via reverse TCA cycle. Both pathways were also highly transcribed in other hydrogen-rich plumes, including the Von Damm vent field. Carboxydicoccus profundi reached up to 4% relative abundance (1.0 x 103 cell ml- 1) in Irinovskoe non-buoyant plume and was also abundant in non-hydrothermally influenced deep-sea metagenomes (up to 5 RPKM). Therefore, CO, which is probably not sourced from the hydrothermal fluids (1.9–5.8 μM), but rather from biological activities within the rising fluid, may serve as a significant energy source in hydrothermal plumes. Taken together, this study sheds light on the chemolithoautotrophic potential of the bacterial community in Mid-Atlantic Ridge plumes.

contamination by gas bubbles.Samples were analyzed for content of 3 He and 4 He isotopes at the mass spectrometric facility of the University of Bremen (Germany) with a combination of a highresolution sector field mass spectrometer and a quadrupole mass spectrometer (1).Total errors based on the instrument performance and standard reference samples for 3 He, 4 He, and Ne concentrations were below 2%, 1%, and 1%, respectively.Hydrothermally sourced primordial He is characterized by an excess of the isotope 3He.Thus, values are reported as δ3 He (in %), defined as (3He/4He)/Ra -1, the fractional deviation of the 3He/4He ratio in samples from that of air (Ra = 1.384 × 10−6).

Thermodynamic calculations
Standard Gibb's energy (∆rGo) of aerobic H 2 and H 2 S oxidation was calculated using SUPCRT92 for a reference state of 2.5°C and 30 MPa.The calculations of ∆rG were done as a function of plume dilutions (up to 1:10 6 fluid:seawater) (Table 3).Constant activities of O 2 (aq): 250µM, sulfate: 3 mM, and H + : pH of 8.0 were assumed.
A new probe was designed in order to visualize and enumerate the SAR202 genus UBA11650.

MAG analysis
The estimation of MAGs' contamination and completeness was done by CheckM v2 (11).
Additionally, the abundance of MAGs was assessed for Irinovskoe and Semenov-2 metagenomes through read recruitment using BBMap (99% identity) (12), and subsequent conversion of values to reads per kilobase per million (RPKM).
A phylogenetic tree was also constructed using translated genes encoding the CODH large subunit gene (CoxL) retrieved from SAR202 MAG_103_1 and MAG_103_2 and 63 sequences of the UniProtKB database (22).Sequences were aligned using MUSCLE (23) and the tree was calculated with FastTree (24).

Single-nucleotide variation calculations
Synonymous (S) and nonsynonymous (N) mutations in MAG_103_1 and MAG_103_2 were determined through SNV patterns in plume and background metagenomes (2000-3000 m depth).
SNV counts and N/S SNV of MAG_103_1 and MAG_103_2 were higher in plumes than in the deep-sea metagenomes.N/S SNVs were <1, indicating that the genes were under stabilizing conditions and mutations were biased toward the synonymous types (Table S7).

Taxonomic descriptions
New taxon has been described under the SeqCode ( 25

Supplementary tables
Table S1: Metagenomic information on three Irinovskoe samples and one Semenov-2 sample.Genomes estimation (GE) was determined by calculating the average sequencing depth of 16 universal, single-copy ribosomal protein genes per sample, yielding counts on a "per genome" basis following Microbe Census (26).Samples were sequenced using the Illumina HiSeq2500 technology.

Table S2 : List of metagenomes and metatranscriptomes recruited onto MAGs retrieved in this study
.BBMap (12)was used with 99% identity filter for metagenomes and 97% identity filter for metatranscriptomes.

Table S3 :
MAGs retrieved in this study.Quality of the MAGs was assessed based on completeness and contamination.MAGs were dereplicated with >95% average nucleotide identity (ANI), with the exception of Sulfurimonas, Sulfurovum and SAR202 MAGs.Abundance of MAGs which share >95% ANI has given only for the most complete MAG.Relative abundance of MAGs in each metagenome was evaluated using a 99% identity threshold with BBMap.MAGs were taxonomically classified using GTDB-tk.

Table S4 : Comparison of the abundance of MAG 65_1 and Thioglobus vulcanius (20) in plumes of Brothers and Macauley vents. Abundance
is given in RPKM.